Transcriptional Regulation Mechanism Of β-fructofuranosidase Gene Expression In The Silkworm Bombyx Mori

Sucrose hydrolases can be divided into α-glucosidase(EC 3.2.1.20)andβ-fructofuranosidase(β-FFase,EC 3.2.1.26)according to enzyme hydrolyzing α-glucosyl or β-fructosyl residue of the substrate,in which the former exists commonly in animals,plants and microorganisms while β-FFase is rarely found in animals.BmSuc1 encoded theβ-FFase of B.mori that was firstly found and cloned from an animal.The latex of mulberry leaves contains high concentration of alkaloidal sugar mimics such as DNJ and D-AB1 which are effective inhibitors of α-glucosidases and highly toxic to some insects that do not normally feed on it,but have no inhibitory effect on β-FFase.As a model lepidopteran insect,the silkworm Bombyx mori is a mulberry-feeding specialist.Our previous research results showed BmSUC1 played an important role in the physiological process of larvae’s normal growth and development by absorbing sucrose nutrients from mulberry leaves.BmSuc1 had tissue specificity for expression in the midgut,anterior and middle silk glands of silkworm larvae.Although the β-FFase gene of a few insects has been identified following BmSuc1,by far,there is a lack of knowledge about the transcriptional regulation mechanism of such newly discovered animal genes.In this study,the truncated fragments of BmSuc1 gene promoter region were constructed,and the core promoter regions were studied by using dual luciferase reporting system.Meanwhile,the potential transcriptional regulators in the core promoter regions and their regulatory effects on BmSuc1 transcriptional expression were also analyzed.The main research results are as follows:1.We first cloned an upstream promoter fragment of about 2 kb of BmSucl and constructed luciferase recombinant reporter vectors with different truncated fragments,which were transfected into silkworm BmN cells to determine luciferase activity.Four core regions on the promoter sequence of BmSuc1 were found---1911 bp to-1820 bp,-1038 bp to-897 bp,-565 bp to-450 bp,and-248 bp to-9 bp.The deletion of these regions would seriously affect the transcriptional activity of BmSuc1.2.By predicting and analyzing the transcription factors in the core promoter regions,we cloned the TATA box-binding protein gene BmTBP.The results of RT-PCR and qRT-PCR showed that BmTBP and BmSuc1 were both highly expressed in midgut and silk gland.In addition,the expression pattern of the two genes in the midgut of B.mori was very similar.This indicated that BmTBP may be involved in the transcriptional regulation of BmSuc1.3.For subsequent studies,we performed prokaryotic expression of BmTBP and purification of recombinant proteins and prepared rabbit polyclonal antibodies.Multiple siRNAs were designed according to the ORF of BmTBP and transfected into BmN cells,in which 61siRNA could significantly reduce the expression of BmTBP in cells at both the transcription level and the protein level.With the decrease of BmTBP expression,the expression of BmSUC1 was also decreased significantly.However,the expression of BmTBP was not affected by the interference of BmSuc1 in the midgut and silk gland of RNAi-BmSuc1 strain.This indicated that BmTBP was probably the upstream regulator of BmSuc1 gene expression.4.Based on the nucleotide sequence of the upstream TATA CRE of BmSuc1,the probe labeled with biotin was designed and used for EMSA experiment with the purified BmTBP protein.The results showed that BmTBP could specifically bind to TATA CRE probe in vitro.Then,ChIP assay was carried out in BmN cells with overexpression of BmTBP,which indicated that BmTBP could also bind to the TATA CRE in cells.These results again suggested that BmTBP could bind to the TATA CRE on the promoter region of BmSucl as an upstream regulator and regulate its expression.5.Another transcription factor--BmTfIIA-S was found by using bioinformatics online analysis tools such as protein interaction,which may interact with BmTBP and participate in the transcriptional regulation of BmSuc1.RT-PCR and qRT-PCR results showed that BmTfIIA-S was highly expressed in the midgut tissue.The results of far-western blot which was performed using purified BmTBP and BmTfIIA-S recombinant proteins showed that they could bind to each other in vitro.The luciferase reporter vectors of BmTBP and BmTflIA-S genes were constructed and co-transfected into BmN cells.We found the promoter activity of BmSuc1 was significantly enhanced.These results indicated that BmTBP could bind to BmTfIIA-S and regulate the transcriptional expression of BmSuc1.6.The possible role of BmSuc1 in the regulation of silk protein expression of B.mori was preliminarily explored.We found it had coordinated effect on the expression of silk gland-specific transcription factors SGF1 and SGF3,while interference of BmSucl did not affect the transcriptional level of Ser1 which was regulated by SGF1 and SGF3.We hypothesized high expression of BmSuc1 in the silk gland may affect post-translational glycosylation of the Ser1 protein.Further research is needed to determine how to modify and the biological significance of the coordination between BmSuc1,SGF1 and SGF3.